Novel Bi-heterocycles as Potent Inhibitors of Urease and Less Cytotoxic Agents: 3-({5-((2-Amino-1,3-thiazol-4-yl)methyl)-1,3,4-oxadiazol-2-yl}sulfanyl)-N-(un/substituted-phenyl)propanamides

The synthesis of a novel series of bi-heterocyclic propanamides, 7a-l, was accomplished by S-substitution of 5-[(2-amino-1,3-thiazol-4-yl)methyl]-1,3,4-oxadiazol-2-thiol (3). The synthesis was initiated from ethyl 2-(2-amino-1,3-thiazol-4-yl)acetate (1) which was converted to corresponding hydrazide, 2, by hydrazine hydrate in methanol. The refluxing of hydrazide, 2, with carbon disulfide in basic medium, resulted in 5-[(2-amino-1,3-thiazol-4-yl)methyl]-1,3,4-oxadiazol-2-thiol (3). A series of electrophiles, 6a-l, was synthesized by stirring un/substituted anilines (4a-l) with 3-bromopropanoyl chloride (5) in a basic aqueous medium. Finally, the targeted compounds, 7a-l, were acquired by stirring 3 with newly synthesized electrophiles, 6a-l, in DMF using LiH as a base and an activator. The structures of these bi-heterocyclic propanamides were confirmed through spectroscopic techniques, such as IR, 1H-NMR, 13C-NMR, and EI-MS. These molecules were tested for their urease inhibitory potential, whereby, the whole series exhibited very promising activity against this enzyme. Their cytotoxic behavior was ascertained through hemolysis and it was observed that all these were less cytotoxic agents. The in-silico molecular docking analysis of these molecules was also in full agreement with their in-vitro enzyme inhibition data.


Introduction
Heterocyclic compounds have been under investigation for a long time because of their important pharmacological properties (1).
Thiazole is one such important heterocyclic system with pronounced pharmacological activities (2). Thiazole is classified under fivemembered heterocyclic class of compounds and is found in many natural and synthetic agents. Naturally, thiazole is available in a large number of terrestrial and marine compounds with different pharmacological activities. Thiazole is also present in the vitamin B1 (Thiamine). In synthetic substituted thiazole derivatives, 2-aminothiazoles have shown a variety of biological activities such as antibacterial, antifungal, antitubercular, anti-HIV, anti-inflammatory, anticancer, anticonvulsant, antidiabetic, antihypertensive, antiprotozoal, dopaminergic, plasminogen activator inhibitor-1, neuroprotective, and antioxidant. This broad spectrum of activities makes 2-aminothiazole as an attractive moiety in medicinal chemistry (3,4).
1,3,4-Oxadiazole is another important heterocycle and its different derivatives possess an extensive spectrum of pharmacological activities such as antiviral, antibacterial, antitumor, antituberculosis, anti-inflammatory, anticonvulsant, and anti-Alzheimer activities (5−11). There have been much advancement regarding synthesis and investigation of biological activities of 1,3,4-oxadiazole derivatives in the last two decades. Several methods have been reported for the synthesis of 1,3,4-oxadiazoles such as reaction of acyl hydrazines with isothiocyanates, reaction of acid hydrazides with carbon disulfide in basic medium, cyclodehydration reaction of diacylhydrazines, reaction of hydrazides with orthoesters, cyclization oxidative reaction of N-acyl hydrazones, and reaction of N-acylbenzotriazoles with acyl hydrazides (12−19).
Urease is known to be involved in different pathogenic processes. It has been known to be involved in pyelonephritis, peptic ulceration, kidney stone, hepatic encephalopathy, urolithiasis, and urinary catheter incrustation (20,21). The molecular docking analysis approximates the ligands regarding their orientation and conformation at binding site of target protein. The precise forecast of activity and precise structural modeling can be achieved by the docking studies (22).
One of the key objectives of organic and medicinal chemists is to design and synthesize the molecules having potent therapeutic values. The rapid development of resistance to existing drugs generates a serious challenge to the scientific community. Consequently, there is a vital need for the development of new drugs having potent activity. The rationale in the present study was that minor modification in the structure of such heterocycles can lead to quantitative as well as qualitative changes in their biological activity. So, in continuation of our previous effort to explore the enzyme inhibitory activity of related bi-heterocyclic bi-amides (23), hereby, we report the synthesis of some novel bi-heterocyclic propanamides as potent urease inhibitors with mild cytotoxicity.

Chemistry
All the chemicals, along with analytical grade solvents, were purchased from Sigma-Aldrich, Alfa Aesar (Germany), or Merck through local suppliers. Pre-coated silica gel Al-plates were used for TLC with ethyl acetate and n-hexane as solvent system (25:75). The spots were detected by UV 254 . Gallonkamp apparatus was used to detect melting points (uncorrected) in capillary tubes. IR spectra (ν, cm -1 ) were recorded by KBr pellet method in the Jasco-320-A spectrophotometer. EI-MS spectra were measured on a JEOL JMS-600H instrument with data processing system. 1 H-NMR spectra (δ, ppm) were recorded at 600 MHz ( 13 C-NMR spectra, at 150 MHz) in DMSO-d 6 using the Bruker Advance III 600 As-cend spectrometer using BBO probe. The coupling constant (J) is given in Hz and chemical shift (δ ) in ppm. The abbreviations used in interpretation of 1 H NMR spectra are as follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; br.t, broad triplet; q, quartet; quint, quintet; sex, sextet; sep, septet; m, multiplet; dist., distorted.

Synthesis of 3-bromo-N-(un/substitutedphenyl)propanamides (6a-l)
The un/substituted anilines (4a-l; 0.038 mol) were suspended in 30 mL distilled water in an iodine flask (100 mL) and aqueous Na 2 CO 3 solution (10%, 2-3 mL) was added. 3-Bromopropanoyl chloride (5; 0.038 mol) was added gradually with vigorous manual shaking. Then this mixture was set to stir on magnetic stirrer for 2-3 h. Reaction completion was monitored by TLC. On completion, the excess ice-cold distilled water (60 mL) was added and the resulting precipitates were collected through filtration, washed with distilled water, and dried to get purified electrophiles, 6a-l.
General procedure for the synthesis of was dissolved in N,N-dimethyl formamide (DMF, 5-10 mL) in a 100 mL RB flask. Solid LiH (0.005 g) was added and the mixture was stirred for half an hour. Then, different aforementioned electrophiles, 3-bromo-N-(un/ substituted-phenyl)propanamides (6a-l; 0.467 mmol), were added and the mixture was set to stirring for 3-5 h. The progress of reaction was monitored through TLC using n-hexane and ethyl acetate solvent system (80:20). On completion, excess ice-cold distilled water was added and the precipitates obtained were filtered, washed with distilled water, and dried to acquire purified products, 7a-l.

Urease inhibition assay
This enzyme assay is the customized form of the commonly known Berthelot assay (23,24). The assay mixture of 85 µL is prepared containing 10 µL of phosphate buffer of pH 7.0 (in each well in the 96-well plate), 10 µL of sample solution and 25 µL of enzyme solution (0.135 units). The contents were preincubated at 37 ºC for 5 min. 40 µL of urea stock solution (20 mM) was added to each well with incubation for 10 min at 37 ºC. It is followed by the addition of 115 µL phenol hypochlorite reagents (freshly prepared by mixing 45 µL phenol with 70 µL of alkali) per well. For color development, incubation was carried out for further 10 min at 37 ºC. The absorbance was measured at 625 nm. The percentage enzyme inhibition and IC 50 values were calculated using the following formula: Where, Control is the total enzyme activity without inhibitor and Test is the activity in the presence of test compound. IC 50 values were calculated using the EZ-Fit Enzyme kinetics software (Perrella Scientific Inc. Amherst, US).

Statistical analysis
Statistical analysis was performed by Microsoft Excel 2010 for all the thrice measured values and the results are presented as mean ± SEM.

Hemolytic activity
Bovine blood samples was collected in EDTA, that was diluted with saline (0.9% NaCl), and centrifuge at 1000 ×g for 10 min. The erythrocytes separated diluted in phosphate buffer saline of pH 7.4 and a suspension was made. Add 20 µL of synthetic compounds solution (10 mg/mL) in 180 µL of RBCs suspension and incubate for 30 min at room temperature. PBS was used as negative control and Triton 100-X was taken as positive control (25,26). The percentage of hemolysis was taken as by using Equation: Hemolysis (%) =

( )
Absorbance of sample Absorbance of negative control Hemolysis % 100 Absorbance of positive control − ×

Molecular docking
Initially, the synthesized chemical ligands (7a-l) were drawn in ACD/ChemSketch tool and retrieved in mol format. Furthermore, UCSF Chimera 1.10.1 tool was employed for energy minimization having default parameters such as steepest descent steps 100 with step size 0.02 (Å), conjugate gradient steps 100 with step size 0.02 (Å) and update interval was fixed at 10. Finally, Gasteiger charges were assigned in ligands using Dock Prep to obtain the good structure conformation. Molecular docking experiment was employed on 7a-l, against Jack bean urease by using virtual screening tool PyRx with VINA Wizard approach (27). The grid box parameters values in VINA search space (X = 11.06, Y = -54.61 and Z = -27.12) were adjusted with default exhaustiveness value = 8 to maximize the binding conformational analysis. We have adjusted sufficient grid box size on biding pocket residues to allow the ligand to move freely in the search space. The generated docked complexes were evaluated on the basis of lowest binding energy (kcal/ mol) values and binding interaction pattern between ligands and receptor. The graphical depictions of all the docked complexes were accomplished by UCSF Chimera 1.10.1 (28) and Discovery Studio (2.1.0), respectively.

Chemistry
The bi-heterocyclic propanamides were synthesized through a protocol depicted in Scheme 1 and different varying groups are listed in Table 1. The molecules were screened against urease to ascertain their enzyme inhibitory potential. The percent inhibition and IC 50 values are given in Table  2. The cytotoxicity of these compounds was also evaluated through hemolytic activity and the results of percentage hemolysis are also tabulated in Table 2.
The structural characterization of one the compounds is discussed hereby in detail for the benefit of the reader. Compound 7a was obtained as a light brown solid with 89% yield. Its molecular formula, C 15 H 15 N 5 O 2 S 2 , was recognized through molecular ion peak at m/z 361 in its EI-MS spectrum. Counting the number of protons in its 1 H-NMR spectrum ( Figure 1A) and number of carbon resonances in its 13 C-spectrum was also supportive in assigning its molecular formula.   Triton-X 89.00 IC50 values (concentration at which there is 50% enzyme inhibition) of the compounds were calculated from the inhibition data obtained after doing assays at high dilutions of the compounds as given in assay method and data was computed using EZ-Fit Enzyme kinetics software (Perrella Scientific Inc. Amherst, USA). Data is mean of three values (mean ± SEM, n = 3). PBS Hemolysis = 2.93%.    Figure 1B). 2-Amino-1,3-thiazol-4-yl heterocyclic moiety was rationalized by two singlets at δ 7.01 (s, 2H, -NH 2 ), and 6.42 (s, 1H, H-5) while the propanamide unit in the molecule was specified by a downfield amidic singlet at δ 10.06 (s, 1H, -NH-CO-1'') along with two broad-triplets in aliphatic region at δ 3.48 (br.t, 2H, J = 6.7, CH 2 -3''), and 2.89 (br.t, 2H, J = 6.7 Hz, CH 2 -2''), symbolic for two interconnected methylenes ( Figure 1C). The signal at δ 4.05 (br.s, 2H, CH 2 -6), was assignable to a methylene uniting the two heterocycles (2-amino-1,3-thiazol-4-yl and 1,3,4-oxadiazole) in the molecule.

Urease inhibition and structure-activity relationship
The synthesized bi-heterocyclic propanamides were screened against urease and found to have potent inhibitory potential against this enzyme, evident from their much lower IC 50 (mM) values as compared to the standard thiourea, as tabulated in Table 2. These derivatives demonstrated inhibition in the range of 5.18 ± 0.06 to 1.24 ± 0.01 µM, relative to thiourea having IC 50 value of 21.11 ± 0.12 µM. So, it was pertinent to say that these molecules possessed many-folds better inhibitory potentials as compared to the standard.
Although the displayed activity is an attribute of a whole molecule, but a limited structure-activity relationship (SAR) was recognized by examining the effect of different groups (-R 1 & -R 2 ) attached to phenyl ring (aryl part) on the inhibitory potential. Figure 4 exposed the general structural features of the studied multifunctional compounds.
Compound 7a (IC 50 = 2.45 ± 0.04 µM) with unsubstituted phenyl ring (aryl part) exhibited very resembling inhibitory potential with mono-substituted molecule 7b (IC 50 = 2.64 ± 0.03 µM) in which a small sized methyl group was present at 2-position. Compound 7c (IC 50 = 1.32 ± 0.02 µM) with methyl group at 3-position, and 7d (IC 50 = 1.75 ± 0.01 µM) with a methyl group at 4-position; however exhibited somewhat greater inhibitory potential relative to 7a. Indeed, the 7c was identified as a second most active compound in the synthetic series. So it was cogent from the present observations that when a small sized group was present at meta or paraposition, the molecule was attributed with superb inhibitory potential ( Figure 5).
However, among the di-methylated regioisomers, compound 7e in which two small sized groups were present at 2-and 4-position, (C)     possessed a remarkable inhibitory potential (IC 50 = 1.24 ± 0.01 µM) and was the most active in synthesized series. A decrease in inhibitory potential (IC 50 = 5.18 ± 0.06 µM) was observed in 7f in which the methyl groups were present at 2-and 5-position as compared to the isomer 7g (IC 50 = 2.63 ± 0.03 µM) in which these groups were present at 2-and 6-position ( Figure 6). It means the presence of one of the methyl group on para-position in aryl part of such regio-isomers, probably resulted in the superb interaction with the active site of the enzyme.
When the inhibitory potential of monoortho substituted molecule 7b (IC 50 = 2.64 ± 0.03 µM) was compared with di-ortho substituted molecule 7g (IC 50 = 2.63 ± 0.03 µM), it was surprising to know that the additional small sized ortho-substituent in latter molecule was not contributing to vary its inhibitory potential as compared to former molecule in a considerable manner (Figure 7).
Among the following two analogues, 7h bearing 3,4-dimethyl residues in aryl part displayed slightly better inhibitory potential (IC 50 = 2.56 ± 0.02 µM) as compared to that of 7i (IC 50 = 3.62 ± 0.01 µM), in which these methyl groups were present at 3-and 5-position ( Figure 8). So it was guessed that the presence of one of the substituent again at para-position attributed the molecule to have suitable interactions with the enzyme.   Instead of small sized groups, when medium sized groups were present in the following three compounds (Figure 9), although their inhibitory potentials were very close, yet a reverse trend was observed on a closer look. Hereby, 7j with ortho-ethyl group behaved as slightly better inhibitor (IC 50 = 2.13 ± 0.01 µM) as compared to both parasubstituted molecules, 7k (IC 50 = 2.85 ± 0.05 µM) and 7l (IC 50 = 2.17 ± 0.02 µM).
So, it was postulated, from the structureactivity relationship among these biheterocyclic propanamides, that the compound bearing a small sized group at meta-position or the di-substituted molecules, bearing at least one small sized group at para-position, were generally potent inhibitors of the urease enzyme. However, when a medium sized group was present on aryl part of these molecules, no significant variation in their   inhibitory potential was observed. Synthesized compounds can be arranged in the following row according to their inhibitory activity: Table 2 for IC 50 values).

Hemolytic activity
The cytotoxicity of the synthesized compounds was evaluated through hemolytic assay. Results of percentage hemolysis are shown in Table 2 which indicated that all these compounds were approximately nontoxic for membrane of red blood cells and their hemolysis values ranged from 5.16% to 11.47%, which were much lower than the Triton-X (positive control) having percentage hemolysis of 89%.

Molecular docking analysis Docking energy evaluation of ligands
To predict the best conformational position within the active region of urease the generated docked complexes were examined on the basis of minimum energy values (kcal/ mol) and bonding interaction pattern such as hydrogen and hydrophobic, respectively. Docking results justified that all compounds, 7a-l, depict good energy values (kcal/mol) (  (29). Based on in-vitro analysis 7e showed good enzyme inhibition potential therefore, ligand 7e was selected to check the binding interaction pattern. The 7e-docking complex showed that ligand structure showed its penetration inside the binding pocket. The 2-aminothiazole part of 7e showed its infiltration toward nickel Recp urease_7e -9 Recp urease_7f -9.2 Recp urease_7g -8.5 Recp urease_7h -8 Recp urease_7i -8.7 Recp urease_7j -8.7 Recp urease_7k -8.1 Recp urease_7l -7.9  The closer view of binding pocket which shows the ligand (yellow color) structure and its conformation inside the binding pocket.   metal and adjusted within binding pocket of urease. However, the substituted aryl moiety showed good conformation position in the opening part of binding pocket of target protein ( Figure 10).
Hydrogen bonds analysis between 7e and urease Figure 11 showed the binding interaction pattern of 7e against urease. In detail binding analysis it was observed that 7e forms four hydrogen bonds at different residues of target protein. The free amino group of thiazole ring and nitrogen within the cyclic ring were both prone to make hydrogen bonds. Our results showed that 2-aminothiazole and cyclic nitrogen of 7e forms two hydrogen bonds against Ala440 and Asp633 having 2.17 and 1.90 Å, respectively. The oxygen of amide group also forms couple of hydrogen bonds with Gln635 and Ala636 with bonds length 2.95 and 3.00 Å, respectively. The docking complex binding pocket residues also showed good correlation with published data which strengthened our docking results (30,31).

Conclusion
A series of bi-heterocyclic propanamides was synthesized in appreciable yields. All these molecules demonstrated an excellent inhibitory potential against urease and their invitro inhibitory results were also coherent with in-silico molecular docking outcomes. These molecules were also very mild cytotoxic towards membrane of the red blood cells. Hence, these molecules might be used as safe and promising drug candidates for ureaserelated ailments.